We are developing a portable, credit-card sized microfluidic lab-on-a-chip for rapid point-of-care detection of multiple bacterial and viral targets using oral fluids. In the initial phase of this study we have evaluated a number of oral fluid collectors, and selected one;designed and evaluated 5 types of miniature PCR devices, and selected one;designed and tested several types of valves, and will progress with two, and assessed a variety of means of moving fluids through the chip and focused on two methods. The detection system utilizes Up-converting phosphor Technology (UPT) and an existing reader. Our initial work focused on HIV (an RNA virus) and B. cereus (a DNA bacterium) to establish proof of principle. The technology involves modular components, and can be modified to detect a wide variety of bacteria and viruses, as well as antibodies to those pathogens. In this renewal, we will continue development of an HIV point-of-care device that monitors viral antigen, nucleic acid, and anti-HIV antibodies. Our bacterial studies will be extended to S. pyogenes, the major organism responsible for strep throat, selected since its high prevalence facilitates clinical evaluation. We will develop and optimize a prototype portable device capable of simultaneously detecting bacterial and viral targets within 2 years, and anticipate having a system ready for full FDA compliant clinical trials in 5 years. The research/development plan presented in this proposal will (1) continue to design, test, and optimize the microfluidic chip and processing unit;(2) carry out clinical studies with banked oral and serum samples from HIV+ and HIV- women (WIHS) and oral swabs from S. pyogenes + and - individuals to assess the robustness, specificity, reproducibility and limits of detection of this device;(3) assess several approaches to enhance sensitivity, decrease analysis time, and lead to a second generation device;and (4) perform assay verification and develop a complete product development plan including a design control program. It is important to note that while the plan focuses on HIV and S. pyogenes in oral samples, the platform can easily be converted to look at other types of samples (blood, nasopharyngeal and oral swabs, urine), and other pathogens including emerging infectious agents such as avian H5N1 virus.